Acid-cleavable compound, use in protein conjugates and drug delivery systems

ABSTRACT

Amino-sulfhydryl cross-linking reagents that are cleavable under mildly acidic conditions are disclosed. Also disclosed are methods of making the cross-linkers, as well as methods of using the cross-linkers, e.g., to deliver a biologically active substance across the membranes of selected cells in a heterogeneous cell population; once inside the cell the active substance is released, intact, by the transient, mild acidity of certain cell structures. Finally, a method of characterizing complex multi-chain protein structures is disclosed.

BACKGROUND OF THE INVENTION

This invention was made with Government support. The Government hascertain rights in this invention.

This is a division of application Ser. No. 645,614 filed Aug. 29, 1984.

This invention relates to controlled release of substances having anamino group to a liquid medium, and to compounds used in such a release.

There are numerous situations in which it is desirable to control therelease of amino-group-containing substances to liquid media. By way ofexample, it may be desirable to control the release of anamino-group-containing drug or cytotoxin to a cell population orspecific members of a cell population. It may also be desirable tocontrol cleavage of various cross-linked proteins or peptides, forexample, in analyzing the spatial relationships in a complex of largeamino-group-containing molecules such as peptides or proteins. [The term"peptide" will be used in this application to include proteins, nomatter how large, as well as shorter-chain peptides.]

One specific situation in which controlled release is desirable isdelivering a biologically active compound through the cell membrane toinner cell structures, for example where the compound has a low orreduced effect if trapped in the medium outside the cell membrane but ismore potent once released inside the cell.

It is also desirable to deliver biologically active compounds toselected cells in a heterogeneous cell population. For example, intreating diseased or infected cells such as virus-infected cells ortransformed or malignant cells, it is desirable to deliver cytotoxins tothe diseased or malignant cells but not to normal cells.

One approach disclosed for targeting biologically active compounds tomalignant cells uses an antibody-toxin conjugate. The antibody isspecific for malignant cells and delivers the toxin to them. To beeffective, such systems should deliver the toxin with high selectivityto the target cells without unnecessarily reducing the effectiveness ofthe active substance. These problems are particularly important wherethe goal is destruction of infected or diseased cells in vivo withoutharming normal cells.

Various cleavable bifunctional cross-linking reagents are known.

Lambert et al. (1981) J. Mol. Biol. 149:451-476 and Wang et al. (1974)Isr. J. Chem. 12:375-389 disclose bifunctional cross-linking reagentscontaining a cleavable disulfide bond; the reagents are used tocharacterize biochemical systems.

Lutter et al. (1974) FEBS Letters 48:288-292 disclose bifunctionalcross-linking reagents with a vic-glycol bond that may be cleaved byperiodate oxidation.

Carlsson et al. (1978) Biochem J. 173:723-737 disclose a procedure forforming disulfide bonds between two different proteins using thebifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate.

More specifically, certain monoclonal antibodies, toxins, and conjugatesthereof are known.

Vitetta et al. (1983) Science 219:644-650 and Edwards (1983) Pharmacol.Ther. 23:147-177 disclose disulfide-linked conjugates of toxins andmonoclonal antibodies specific to cell-surface structures; theseconjugates are used to target toxins toward specific cells havingsurface structures recognized by the antibodies.

Ramakrishnan et al. (1984) Cancer Research 44:1398-1404 discloseconjugating pokeweed anti-viral protein (PAP) to anti-Thy 1.1, amonoclonal antibody. The conjugate is used to inhibit protein synthesisselectively in Thy 1.1-positive target leukemia cells. The linker usedto form the conjugate is N-succinimidyl-3-(2-pyridyldithio) propionate.When the disulfide bond is cleaved, the free PAP toxin is produced.

Ritz et al. (1980) Nature 283:583-585 disclose a monoclonal antibody(J5) that is specific for common acute lymphoblastic leukemia antigen.

Barbieri et al. (1982) Biochem J. 203:55-59 disclose the purificationand partial characterization of an antiviral protein known as pokeweedantiviral protein-S ("PAP-S").

Stirpe et al. (1980) J. Biol. Chem. 255:6947-6953 disclose a method ofpreparing gelonin, a protein cytotoxin.

Neville et al. U.S. Pat. No. 4,359,457 disclose a conjugate of anti-Thy1.2 monoclonal antibody and ricin used as a tumor suppressivecomposition against lymphoma. The linking agent used ism-maleimidobenzoyl-N-hydroxysuccinimide.

The above approaches either depend on the toxicity of an antibody-toxinconjugate, or they depend on disulfide bond cleavage, a phenomenon thatmay be difficult to control temporally and spatially to avoid release ofthe toxin before delivery to the targeted cells.

Kirby et al. (1970) Proc. Biochem. Soc. Symp. 31:99-103 disclose thatmaleic acid amides are rapidly hydrolyzed below pH 3, and thatsubstitution of maleamic acid increases that rate, with a t-butylsubstituent providing the largest increase and a methyl substituent thesmallest.

Dixon et al. (1968) Biochem J. 109:312-314 disclose reversible blockingof amino groups using 2-methylmaleic ("citraconic") anhydride as ablocking reagent. The amide bond between the citraconyl residue and alysine residue of insulin was not cleaved at pH 6.5; when the pH waslowered to 3.5 at 20° C. overnight, there was total release of theblocking group, leaving the insulin unchanged.

SUMMARY OF THE INVENTION

We have discovered a class of cross-linking agents that permitcontrolled release of an amino-group-containing substance under mildlyacidic conditions.

In one aspect, the invention features a cross-linking reagent suitablefor forming an acid-cleavable link between the amino nitrogen of anamino-group-containing substance and a sulfhydryl function of a secondcompound. The cross-linking reagent comprises the unit ##STR1## where R₁and R₂ are independently selected from H and alkyl groups of C₅ or less,and A comprises a bridge unit.

Alternatively, the cross-linking reagent comprises the unit ##STR2##where R₂ is selected from H and alkyl groups of C₅ or less, A comprisesa bridge unit, and s=2-5.

In a second aspect, the invention features the cross-linked heterodimercomplex that results from cross-linking an amino-group-containingcompound with a sulfhydryl-group-containing compound. Theamino-group-containing compound is released from the complex bydecreasing the pH of the medium enough to hydrolyze the amide link toregenerate the amino-group-containing compound.

In preferred embodiments of the first two aspects of the invention, thebridge comprises the unit A, ##STR3## where: R₃ -R₆ are independentlyselected from H, a halogen, alkoxy and alkyl groups of C₅ or less, andtertiary amines; and B is a linker to the maleimido function. R₃ -R₆ andB are selected so that the sulfur attached to the ring of unit A has apK_(a) low enough in the corresponding thiophenol compound such that,during formation of that sulfide group in A, competing side reactionscaused by OH⁻ are avoided; for example, the pK_(a) of the thiophenol isbelow 10.0.

Also in preferred embodiments, the sulfide function of the aromatic ringin A is linked to the maleic acid function by a group comprising(CH₂)_(k) where k is 1-5. Most preferably, B comprises an amide functionwhose nitrogen is linked to the aromatic ring of A para to the sulfidefunction, and whose carbonyl function is linked to the nitrogen of themaleimido group by (CH₂)_(q) [where q=1-5] or by an aryl group [mostpreferably an unsubstituted aryl group in which the maleimido link ismeta or para to the carbonyl link].

Also in preferred embodiments of the resulting cross-linked complex, theamino-group-containing substance is a biologically active substance tobe delivered to selected cells, and the sulfhydryl-group-containingcompound comprises a binding partner that is selective for those cells;the compound is used in a method of delivering the active substance toselected cells as described more fully below in connection with thefourth aspect of the invention.

In a third aspect, the invention features a method of synthesizing thecross-linking agent by a substitution reaction linking an activatedsubstituted maleic acid derivative to a compound comprising athiophenol-containing function; the reaction is performed at a pH highenough to ionize the sulfhydryl group of the thiophenol, therebyavoiding competing SN₂ ' substitution reactions, and low enough to avoidcompeting reactions of OH⁻. The resulting intermediate is then linked toa maleimido function. In preferred embodiments of the method ofsynthesis, the substitution reaction is conducted at a pH between about9.5 and 11.5; most preferably the pH is between 10.5 and 11.5, or about11.0. Also in preferred embodiments, the thiophenol-containing functionincludes an amine that is linked to the maleimido function by an amidebond that includes that amine nitrogen.

In a fourth aspect, the invention features a method of delivering anamino-group-containing biologically active substance to selected membersof a heterogeneous population of cells by exposing the cells to acomplex formed by cross-linking the active substance to asulfhydryl-functionalized cell-binding partner specific for acell-surface receptor of the selected cells. The compound bindsselectively to those cells, and the active substance is released fromthe complex by exposure to a pH low enough to cleave the amide bondbetween the active substance and the maleic acid function of thecross-link.

In preferred embodiments of the fourth aspect, the biologically activesubstance is a peptide or protein, most preferably a cytotoxic substancethat inactivates protein synthesis by inactivating ribosomes. The cellbinding partner is an antibody--for example, a monoclonal antibody, thatrecognizes cell-surface antigens such as J5 antibody or antibodies toT3, T4, T11, or T12 T-cell surface antigens.

Also in the preferred method of delivery, the antibody is one that istransported across the membrane of cells containing the surface antigenso that the cross-linked complex is transferred within the cell. Onceinside the cell, the complex is exposed to a specific cell compartmentthat naturally exhibits acidity sufficient to release the activesubstance, e.g. the ribosome-inactivating toxin, thus delivering itefficiently and selectively.

The invention allows delivery of an amino-group-containing substance toa liquid medium under carefully controlled conditions that are mild inthat they do not require oxidative or reductive conditions or extreme pHvalues that could alter either the amino-group-containing substance orother components of the liquid medium system. Moreover, cleavage occursat a pH that is not only mild enough to be consistent with in vivosystems but is actually triggered by naturally occurring intracellularphenomena. Advantageously, the amino-group-containing substance isreliably released at that pH but is stably bound at higher pH values.Preferably, release of the amino substituent is achieved at pH 3.5-5.5and most preferably at pH 4.0-5.0, or about 4.5. Moreover, thecross-linking and release process neither adds units to nor subtractsunits from the amino-group-containing substance.

Other features and advantages will be apparent from the followingdescription of the preferred embodiments and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

We turn now to a description of the preferred embodiments of theinvention, first briefly describing the drawings thereof.

Drawings

FIG. 1 is a flow diagram of the preparation of a class of cross-linkingreagents.

FIG. 2 is a flow diagram of the preparation of a specific complexcross-linked by the reagents prepared in FIG. 1.

There are two particularly preferred embodiments of the invention. Oneis a cell-delivery agent for delivering a biologically active protein orpeptide to a cell as described above. The other is a tool for evaluatinga complex that includes one or more large amino-group-containingmolecules, such as a ribosome complex or a multi-unit enzyme. Bycross-linking members of the complex, it is possible to separate out thecross-linked components, and then to decouple the cross-linkedcomponents without altering their structure in order to analyze them.Analysis of the appearance and disappearance of members of the complexand of the cross-linked substance provides a tool for evaluatingcomponents of living systems and thereby to follow the effects oftreatments of, and abnormalities in, these systems.

Cell-Delivery Embodiment

The preferred biologically active substance to be delivered to a cell isa protein or peptide drug or an enzyme. In a particularly preferredembodiment, the active substance is a cell toxin to be delivered toselected cells. Such toxins include the pokeweed antiviral proteins PAP,PAPII, and PAP-S, described above and in Irvin (1975) Pharmac. Ther.21:371-387. Other peptide cytotoxins include ricin A-chain, abrinA-chain, modeccin A-chain as well as gelonin and other single-chainribosomal inactivating proteins such as those described in Barbieri etal. (1982) Cancer Surv. 1:489-520.

Peptide toxins are preferred because they are readily bound to thereagent and because they are extremely potent toxins which inhibit cellprotein synthesis when present inside the cell in extremely minutequantities. Other amino-group-containing cytotoxins which are notpeptides are also within the scope of the invention however; examples ofsuch cytotoxins or cytotoxic drugs are melphalan, bleomycin, adriamycin,and daunomycin.

The above-described active substances are delivered to selected cells bybinding partners to cell-surface features. The preferred bindingpartners are antibodies to cell surface antigens. Particularly preferredare monoclonal antibodies to cell surface antigens specific to diseased,infected, transformed or malignant, but not to normal cells.Particularly, but not exclusively, they are antibodies that are taken upby the cells. It is not necessary that normal (i.e., non-target) cellslack the specific antigen entirely, as long as the antigen is notpresent in sufficient numbers on those cells to permit significantuptake of the active substance by the cells.

One such antibody is J5, described above, available from CoulterImmunology, Hialeah Fla. Other examples are antibodies to melanoma,surface antigens such as those described by Imai et al. (1983) CancerImm. 15:206 et seq. Other suitable antibodies available from CoulterImmunology include the antibodies to surface antigens found on T-cellsand T-cell lymphomas such T3, T4, T11, and T12.

Other binding partners in this embodiment include non-antibody cellmembrane transport agents such as transferrin and protein hormones suchas insulin.

Biological System Evaluation

The preferred systems to be evaluated by cross-linking include cellstructures such as ribosomes and membranes, and complex proteins. Onecomponent of the system is functionalized with a sulfhydryl group, andthe above-described cross-linking agent is used to cross-link thatcomponent to an amino-group-containing substance that is adjacent to thesulfhydryl group. For example, a functionalized enzyme substrate may beused to establish the binding domain of the enzyme by cross-linkingduring the progress of the enzyme-catalyzed reaction.

The Cross-Linker

In either of the two above-referenced embodiments, the linker joiningthe amino-group-containing substance and the sulfhydryl group isextremely important. The amino-group-containing substance should bejoined to and released from the linker without alteration of itsactivity and preferably without alteration of its structure.

Thus, the amino-linkage should be formed under mild conditions that donot affect protein structure; at that same time, the linkage should be astrong one that is not cleaved prematurely and yet is cleaved under mildconditions that are compatible with sensitive components of a biologicalsystem. The cleavage site should be at the bond between the amino groupof the amino-group-containing substance and the linker, so that thesubstance is released intact, with no additional component and with nodeletions.

As described above, the maleic anhydride function is appropriate forforming a cleavable amide bond to the nitrogen of theamino-group-containing substance. Also, the maleimido entity is suitablefor forming a stable link to the sulfhydryl function of the secondcompound of the complex.

The bridge between the above-described maleimido entity and maleicanhydride entity is important because if that bridge is cleaved bynatural processes (e.g., by enzymes in an in vivo system) at anundesired time or place, the selectivity of the binding partner is lost,and the toxin, or a compound with potential toxic activity, is presentin the medium outside the cells without a means for selecting the targetcells. Other constraints on the bridge are compatibility and inertnesswith respect to other components of the complex and ease of formationunder conditions that do not harm those components.

The preferred bridges are described above in detail in the Summary ofthe Invention.

For purposes of illustration, we will now describe the synthesis and useof two specific representative examples of the following preferredcross-linking reagents having the formula: ##STR4##

In the first example, W is (CH₂)₅ ; in the second example, W is ##STR5##

Synthesis of the Cross-Link

Synthesis of the first example is illustrated in FIG. 1. In briefoutline, cross-linking reagent 6 can be synthesized by the followingmajor steps:

1. hydrolysis of 2-bromomethyl maleic anhydride (compound 2);

2. reaction of the resulting maleic acid derivative (compound 3) with4-aminothiophenol to give compound 4; and

3. reaction of compound 4 with the acyl chloride of 6-maleimido-caproicacid to yield the diacid which is dehydrated to form compound 6.

Steps are taken to avoid isomerization of 2-(bromomethyl)-maleic acid(3) to the corresponding fumarate analog by hydrolyzing the anhydride(2) in a dilute aqueous solution and neutralizing the diacid (3)immediately after complete hydrolysis. Any bromomethyl fumaric acidderivative formed is removed in purifying compound (4) as described inmore detail below.

Undesired S_(N) 2' substitution reactions (rather than the desired S_(N)2 substitution) of bromomethyl maleic acid are suppressed by raising thepH of the reaction mixture enough to convert the p-aminothiophenol tothe corresponding thiophenolate anion.

Specific details of the above-described synthesis are as follows.

1. 2[2-(4-aminophenyl)-2-thiaethyl]-maleic acid(4)

2-(Bromomethyl)-maleic anhydride (2) is prepared from compound 1according to the method of Laursen et al. (1971) J. Med. Chem.14:619-621 with the modification by Greenlee and Woodward (1980)Tetrahedron 36:3367-3375. This material (2 g) is hydrolyzed in water (20mL) for about 1 hour at room temperature to 2-(bromomethyl)-maleic acid(3) and the solution is neutralized with 1 N NaOH (21.2 mL), degassedand kept under N₂. The solution is added to a previously preparedsolution of 4-aminothiophenol (1.51 g) that has been dissolved indegassed 1 N NaOH (11.6 mL) with gentle heating under N₂ and filtered toremove the insoluble dimer. The filtrate is diluted with degassed water(12 mL) before being added to the above solution of compound 3. Thefinal reaction solution is brought to about pH 11.0 with 1N NaOH andstirred under N₂ at room temperature overnight. The solution is thenfiltered to remove small amounts of bis-(4-aminophenyl)-disulfide,cooled on ice, and acidified with 1N HCl, which gives a yellowprecipitate. n-Butanol is added (40 mL) and the mixture is vigorouslystirred on ice for 5 minutes. This removes the fumaric acid analog ofcompound 4. The solid is then collected by filtration, washedsuccessively with 0.1 N HCl and water, and finally dried in a vacuumdesiccator over P₂ O₅, to yield the neutral compound 4.

2. Maleimido-diacid 5

6-Maleimido-caproic acid is prepared according to the method of Kellerand Rudinger (1974) Helv. Chim. Acta 58:531-541 and transformed to itsacid chloride with thionyl chloride. 6-Maleimido-caproic acid (1.06 g)is dissolved in dry THF (10 mL), treated with SOCl₂ (0.51 mL) and thesolution is refluxed with exclusion of moisture for 1.5 hours. Thesolution is evaporated in vacuo to dryness and the remaining oil isdissolved in dry THF (5 mL). This solution is added dropwise over 10minutes to a solution of compound 4 (1.16 g) in dry THF (15 mL) andN-ethylmorpholine (2.16 mL). After 2 hours, the solution is evaporatedto dryness under reduced pressure. The remaining oil is taken up intoethyl acetate and the solution is washed with cold 0.1 N HCl and thenwith water. The ethyl acetate solution is dried, and the solvent isremoved by evaporation. The crude product is then purified by flashchromatography on silica gel with CHCl₃ --MeOH (95:5, v/v), containing2% acetic acid as eluant. The pure product is finally passed through acolumn of Dowex 50 (H⁺ form) in MeOH--H₂ O (1:2, v/v). Evaporation ofthe solvent and drying of the oily residue under high vacuum gives abrittle solid foam in 55% yield (976 mg).

3. Cross-Linking Reagent 6

Compound 5 (200 mg) in dry THF (3 mL) is treated at 0° C. with asolution of dicyclohexylcarbodiimide (110 mg) in dry THF (2 mL). Thereaction mixture is stirred at room temperature for 45 minutes, and thenfreed from the precipitated dicyclohexylurea by filtration. The filtrateis concentrated in vacuo to dryness and the resulting oil is dissolvedin dry dioxane (2 mL) and filtered again. The dioxane is finally removedby lyophilization, leaving a brownish solid.

The second example of the cross-linking reagent will be prepared in thesame manner as the above-described compound 6 using thep-maleimidobenzoylchloride in place of the 6-maleimidocaproic acidchloride of step 2, above.

Cross-Linking

The two cross-linking agents described above can be used to cross-link apeptide drug or toxin to a cell-specific antibody as described below.The specific example described utilizes the toxin gelonin and themonoclonal antibody J5, described above. FIG. 2 diagrams these steps.

Gelonin is obtained from seeds from Gelonium multiflorum (Euphorbiaceae)as described by Stirpe et al. (1980) cited above. The seeds areavailable from United Chemical and Allied Products, 10 Clive Row,Calcutta-1, India, through the Meer Corporation, North Bergen, N.J. Asample of gelonin (1.47 mg/mL) in 100 mM sodium phosphate buffer, pH7.2, is treated at 20° C. for 30 minutes with a 12-fold excess ofcompound 6 in DMSO. The sample is then applied to a column of SephadexG-25 (superfine) at 4° C. equilibrated with 100 mM sodium phosphatebuffer, pH 7.0, containing EDTA (1 mM) to remove excess compound 6 fromthe newly formed compound 7.

Compound 7 is then utilized to form a cross-link to the monoclonalantibody J5 which has been modified with 2-iminothiolane as describedbelow in order to introduce 2.0 mol sulfhydryl groups per mol antibody.

J5 antibody (2 mg/ml) in 60 mM triethanolamine-HCl buffer, pH 8.0,containing potassium phosphate (7 mM), NaCl (100 mM), EDTA (1 mM), isdegassed and then treated with 2-iminothiolane (1 mM) for 90 minutes at0° C. under nitrogen. Stock solutions of 2-iminothiolane hydrochloride(0.5 M) are prepared as described previously [Lambert et al. (1978)Biochemistry 17:5406-5416]. The reaction is terminated by gel filtrationat 4° C. through a column of Sephadex G-25 (fine) equilibrated with 5 mMbistris-acetate buffer, pH 5.8, containing NaCl (50 mM) and EDTA (1 mM).Sulfhydryl groups introduced into the antibody in this way arequantified spectrophotometrically by the method of Ellman (1959) Arch.Biochem. Biophys. 82:70-77.

As described below, this derivatized antibody is mixed with a 5-foldmolar excess of compound 7 that has a level of maleimide substitution of0.7 groups per mol gelonin. The higher level of substitution of J5increases the yield with respect to the antibody while the low level ofsubstitution of gelonin reduces the amount of high molecular weightaggregates formed in the cross-linking reaction mixture. The final yieldof conjugate after gel filtration and carboxymethyl cellulosepurification is 37% with respect to J5.

Modified J5 (8 mg, 0.05 μmol) in 5 mM bistris-acetate buffer, pH 5.8 (15mL), containing NaCl (50 mM), and EDTA (1 mM) is mixed at 0° C. with a5-fold molar excess of complex 7 (8 mg of gelonin) in 100 mM sodiumphosphate buffer, pH 7.0 (11 mL), containing EDTA (1 mM) and then with0.5 M triethanolamine-HCl buffer, pH 8.0, (0.15 mL) to give a final pHof 7.0. The mixture is incubated at 0° C. for 2 hours and then freshlyprepared N-ethylmaleimide (1 mM) in ethanol (0.26 mL) is added to blockany remaining free sulfhydryl groups. After 30 minutes at 0° C., thesolution is maintained on ice while concentrating to 13 mL using animmersible ultrafiltration unit (Millipore Corporation, CX-10 filter).The mixture is then applied to a column (95 cm×2.6 cm) of SephacrylS-300 equilibrated at 4° C. with 5 mM sodium phosphate buffer, pH 7.0,containing NaCl (15 mM), and NaN₃ (0.4 mM). Gel filtration separates theconjugate and native J5 (M_(r) =160,000) from non-cross-linked gelonin[M_(r) =30,500; Thorpe et al., (1981) Euro. J. Biochem. 116:447-454],and from some high molecular weight aggregates. The major peak,corresponding to a molecular weight range of 160,000 to 220,000 andshown by polyacrylamide/sodium dodecyl sulfate gel electrophoresis tocontain both native J5 and cross-linked conjugate 8 is pooled and passedthrough a column (5 mL bed volume) of carboxymethyl cellulose (WhatmanCM-52) equilibrated in the same buffer. The column is washed with 1column volume of buffer and the eluants combined. Under these preciseconditions of ionic strength and pH, native J5 is bound by the columnwhile cross-linked complexes of type 8 pass through without retention.Monoclonal antibodies are heterogeneous with respect to charge [Cowan etal. (1973) FEBS Lett. 30:343-346]: the sample of J5 used in theseexperiments is, prior to modification, passed through a carboxymethylcellulose column under identical conditions. Only the J5 that bound tothe column is used in cross-linking experiments. It is eluted from thecolumn with buffer containing 1.0 M NaCl (yield 66%).

The solution containing purified complex 8, separated from thenon-cross-linked monomeric proteins, is concentrated at 0° C. using animmersible ultrafiltration membrane (Millipore, CX-30), dialysed against10 mM potassium phosphate buffer, pH 7.8, containing triethanolamine(0.5 mM), and NaCl (145 mM), and finally stored at 4° C. aftersterile-filtration through a Millex-GV filtration membrane (0.22 μm,Millipore).

Acid Cleavage

The purified complex 8, or a corresponding complex prepared using thesecond cross-linker example described above at page 11, can beadministered to a population of cells that includes normal cells andcommon acute lymphoblastic leukemia cells that exhibit a surface antigenrecognized by J5 antibody. The J5 component in the complex retains itsability to bind selectively to cells with that surface antigen, asjudged by indirect immunofluorescence on Namalwa cells as described byRitz et al. (1980) J. Immunol. 125:1506-1514. The gelonin component ofcomplex 8 exhibits reduced ability to stop protein synthesis incomparison to native, non-cross-linked gelonin, as determined by therabbit reticulocyte lysate system such as the system available from NewEngland Nuclear Company.

Thus, the complex has cell-recognition capability, and once the antibodybinds to the cell, the complex is internalized into a cell compartmentwhere cleavage occurs. Specifically, receptors which are internalized byreceptor-mediated endocytosis pass through acidified compartments knownas endosomes or receptosomes [de Duve (1983) Eur. J. Biochem.137:391-397]. Thus, the complex will be exposed transiently to an acidicpH sufficient to cleave the active amino-group-containing substance.

Cleavage of native gelonin from complex 8 is relatively rapid with goodyields below about pH 5. For example, at 37° C. and pH 4, over 30% ofthe total gelonin is cleaved by 5 hours, and over 50% is cleaved after10 hours. At pH 5, about 15% is cleaved after 5 hours and about 25% iscleaved after 10 hours. Above pH 6, the amide link to gelonin isrelatively stable.

For a given drug-antibody complex, one skilled in the art will recognizethat the yields and release rates can be measured for the pH andtemperature conditions that are experienced in the release environment,and on that basis the amount of complex necessary to provide sufficientactive substance to the target cells can be determined. For example,drug release can be determined using polyacrylamide/sodium dodecylsulfate gel electrophoresis.

A method for testing the release rate of gelonin under varying pHconditions is exemplified by the following procedure.

Samples of 8 (0.54 mg/mL) are diluted 1:2 (v/v) with 100 mM citricacid--phosphate buffer to give final pH values of 4.0, 5.0, and 6.0. Thesolutions are incubated at 37° C. and samples withdrawn at differenttimes for analysis of gelonin activity, and for analysis bypolyacrylamide/sodium dodecyl sulfate gel electrophoresis. Samples forthe former are diluted 10-fold into 150 mM Tris-HCl buffer, pH 8.8,containing sodium phosphate (9 mM), NaCl (18 mM) and bovine serumalbumin (0.2 mg/mL) and are then stored at 4° C. prior to assay; thefinal pH is 8.5. Samples (50 μL) for polyacrylamide gel analysis areplaced on VSWP-025 dialysis membranes (Millipore) floating on 5 mMtriethanolamine-HCl buffer, pH 7.8, containing potassium phosphate (5mM) and NaCl (70 mM), and dialysed for 2 hours at 4° C. after which thepH of the samples is 7.8.

Characterizing Protein Complexes

Finally, the cross-linking reagent 6 can be used as described by Lambertet al. (1981) J. Mol. Biol. 149:451-476; Wang et al. (1974) Isr. J.Chem. 12:375-389; Lomant et al. (1976) J. Mol. Biol. 104:243-261; Lutteret al. (1974) FEBS Letters 48:288-292; or Coggins et al. (1976)Biochemistry 15:2527-2532 to analyze complex structures containing manypolypeptide chains.

A chain of interest is functionalized with a sulfhydryl group andexposed to the cross-linking reagent 6. An amino group of an adjacentchain will form the cleavable amide link at pH 7 or above. Hydrolysis ofthe amide link at a pH that does not denature the chains (e.g. pH 4 to5) can be followed, e.g. by SDS gel electrophoresis to identify thechains adjacent to the chain of interest. The ability to controlhydrolysis under non-denaturing conditions, non-oxidative, andnon-reducing conditions is particularly important. It is also importantto be able to form the cross-link to any amino-containing chain site,and not merely to rely upon the availability of free sulfhydryl groupswhich are inside the molecule.

Other Embodiments

Other embodiments are within the following claims. For example, otherdrug-delivery complexes and protein-evaluation cross-linkers are withinthe claims.

We claim:
 1. An acid-cleavable complex for controlled release of anamino-group-containing substance to a liquid medium, said complexcomprisingsaid amino-group-containing substance, a sulfide-linkedprotein, and an acid cleavable linker between the sulfide-linked proteinand the amino-group-containing substance, said complex comprising one ofthe two following units: ##STR6## where: one of X and Y is O⁻ ; theother of X and Y is the residue of the amino-group-containing substancewhose amino nitrogen forms an amide link with a carbonyl group of themaleic acid function; Z is the sulfide-linked protein, including thesulfur of that link; R₁ and R₂ are independently selected from H andalkyl group of C₅ or less; and A is a bridge unit; and ##STR7## where X,Y, Z, R₂, and A are as defined above, and s=2-5.
 2. The complex of claim1 wherein A comprises the unit ##STR8## where R₃, R₄, R₅, and R₆ areindependently selected from H, halogens, alkoxy and alkyl groups of C₅or less, and tertiary amines; and B is a linker to the maleimidofunction.
 3. The complex of claim 2 wherein R₃, R₄, R₅, R₆, and B areselected so that the sulfur attached to the aromatic ring of A haspK_(a) low enough in the corresponding thiophenol such that duringformation of the sulfide group in A, competing reactions of OH⁻ areavoided.
 4. The complex of claim 1 wherein the protein comprises anantibody.
 5. An acid-cleavable complex for controlled release of abiologically active amino-group-containing substance to a liquid medium,said complex comprisingsaid amino-group-containing substance, asulfide-linked substance comprising a cell binding partner for a cellsurface receptor, andan acid cleavable linker between the sulfide-linkedsubstance and the amino-group-containing substance, said complexcomprising one of the two following units: ##STR9## where: one of X andY is O⁻ ; the other of X and Y is the residue of theamino-group-containing substance whose amino nitrogen forms an amidelink with a carbonyl group of the maleic acid function; Z is thesulfide-linked substance, including the sulfur of that link; R₁ and R₂are independently selected from H and alkyl groups of C₅ or less; and Ais a bridge unit; and ##STR10## where X, Y, Z, R₂, and A are as definedabove, and s=2-5.
 6. The complex of claim 5 wherein A comprises the unit##STR11## where R₃, R₄, R₅, and R₆ are independently selected from H,halogens, alkoxy and alkyl groups of C₅ or less, and tertiary amines;and B is a linker to the maleimido function.
 7. The complex of claim 6wherein R₃, R₄, R₅, R₆, and B are selected so that the sulfur attachedto the aromatic ring of A has a pK_(a) low enough in the correspondingthiophenol such that during formation of the sulfide group in A,competing reactions of OH⁻ are avoided.
 8. The complex of claim 5wherein said cell-surface receptor is an antigen and said bindingpartner is an antibody to that antigen which is transported across cellmembranes containing said receptor,whereby the complex is capable ofbinding to the receptor and of being transported across the membrane,and the biologically active substance is released when the pH of thelocal intracellular environment is decreased.
 9. The complex of claim 5wherein said biologically active substance is a peptide.
 10. The complexof claim 5 wherein said biologically active substance is cytotoxic. 11.The complex of claim 10 wherein said cytotoxic substance is a proteinsynthesis inhibitor.
 12. The complex of claim 11 wherein said cytotoxincomprises a ribosomal inactivating protein.
 13. The complex of claim 12wherein said cytotoxin comprises a pokeweed antiviral protein, gelonin,the ricin A-chain, abrin A-chain, or modeccin A-chain.
 14. The complexof claim 5 wherein said cell binding partner is an antibody to a T-cellsurface antigen.
 15. The complex of claim 14 wherein said T-cell surfaceantigen is T3, T4, T11, or T12.
 16. The complex of claim 5 wherein saidcell binding partner is an antibody to a tumor-associated surfaceantigen.
 17. The complex of claim 5 wherein said cell binding partner isJ5 antibody.